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May 25, 2012 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-12 

TO: Biologics Licensees, Permittees, and Applicants 

 Veterinary Services Management Team 

 Directors, Center for Veterinary Biologics 

 Deputy Administrator, Animal Care 

FROM: Richard E. Hill, Jr. /s/ Richard E. Hill, Jr. 

 Director 

 Center for Veterinary Biologics 

SUBJECT: Use of Humane Endpoints and Methods in Animal Testing of Biological 

 Products 

I. PURPOSE 

This Notice informs licensees, permittees, and applicants of current Center for Veterinary 

Biologics (CVB) policy concerning the use of humane endpoints in animal challenge 

tests and the use of anesthesia before and during intracerebral (IC) inoculation. It 

confirms CVB’s commitment to replacing, reducing, and refining animal use. 

II. BACKGROUND 

Title 9, Code of Federal Regulations (9 CFR), Part 117.4(e) indicates that animals used in 

testing of biological products may be treated or humanely destroyed if illness has 

progressed to a point where death is certain to occur. The definition of that point (the 

humane endpoint) is to be specified in the Outline of Production. This Notice clarifies 

the humane endpoint for all animal challenge potency tests codified in 9 CFR 113, 

provides guidance on establishing humane endpoints for potency tests that do not follow 

those standard requirements, and provides for the use of anesthesia for IC inoculation of 

mice for inactivation and potency testing. 

III. POLICY 

All codified potency tests (with the exception of the rabies challenge test discussed 

below) that are conducted by administering viable virus, bacteria, or a bacterial toxin to 

animals in a dose that is expected to be lethal should be modified (in the Outline of 

Production) to include the following wording, unless justification for alternative wording 

acceptable to the CVB is provided: 

Moribund animals exhibiting clinical signs consistent with the expected disease 

pathogenesis that are unable to rise or move under their own power should be 

United States 

Department of 

Agriculture 

Animal and Plant 

Health Inspection 

Service 

Veterinary Services 

Center for Veterinary 

Biologics 

1920 Dayton Avenue 

PO Box 844 

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(515) 337- 6100 
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-12 

Page 2 

humanely euthanized and considered as deaths as outlined in 9 CFR 117.4. 

Observations must be made with sufficient frequency to ensure suffering is 

minimized. 

In the case of the rabies challenge described in 9 CFR 113.209, 9 CFR 113.312, and 

Supplemental Assay Method 308, acceptable wording is: 

Animals exhibiting paresis, signs of paralysis, and/or convulsions must be 

humanely euthanized and considered as deaths as outlined in 9 CFR 117.4. 

Similar definitions should be incorporated into non-codified potency and safety tests and 

efficacy study protocols. Proposed endpoints will be reviewed on a case-by-case basis. 

When implementing humane endpoints, equal treatment must be given to both vaccinated 

and control animals. Criteria should be as objective as possible, to minimize differences 

in individual interpretation. Observations must be documented and support euthanasia 

based on humane endpoint criteria. 

Firms are strongly encouraged to use anesthesia for IC inoculation of mice when 

conducting rabies vaccine inactivation testing under 9 CFR 113.209(d)(2)(i) and for IC 

inoculation with challenge virus for potency testing under 9 CFR 113.209(d)(3). Firms 

wishing to use anesthesia in such testing should contact their Reviewer. 

Care and attention, including training and procedures, must be used to ensure that the 

anesthesia is performed in such a way as to minimize pain and suffering. 

The CVB encourages the use of analgesics in animal studies and potency testing, when it 

can be shown this does not affect the study outcome. 

Additional policy and guidance is currently under development. 

IV. IMPLEMENTATION/ APPLICABILITY 

This change is effective six months from the date of this Notice. 

In implementing the requirements of this Notice, firms are strongly encouraged to use the 

following references, as applicable: 

Supplemental Assay Methods, including 308, 613, 624, 625, 626, and 627, 

The references cited on the “Human Endpoints and Euthanasia” website maintained by 

USDA’s Animal Welfare Information Center, and 

Appendix 2 of the Report and Recommendations of the European Centre for the 

Validation of Alternative Methods (ECVAM) Workshop 48.